Low-cost preparation method of high-purity chlorogenic acid in folium cortex eucommiae

Abstract

The invention provides a low-cost preparation method of high-purity chlorogenic acid in folium cortex eucommiae, and the low-cost preparation method comprises the steps of ethyl acetate two-phase solvent extraction, water-based ultrasonic-assisted extraction and solid-phase extraction. Compared with other existing methods, the low-cost preparation method has the characteristics of simplicity, convenience and low cost, and is suitable for large-scale preparation of the high-purity chlorogenic acid in the folium cortex eucommiae.

Description

Technical Field

[0001] The invention belongs to the technical field of biological extraction, and in particular is a low-cost preparation method of high-purity chlorogenic acid in eucommia leaves. Background Art

[0002] Eucommia leaf is the dried leaf of Eucommia ulmoides, which has multiple pharmacological effects, including anti-oxidation, antihypertensive, blood sugar regulation, osteoporosis treatment, etc. It has been listed as a new drug in the Chinese Pharmacopoeia since 2005, and was included in the Catalogue of Medicines and Foods of the Same Origin by the National Health Commission and the State Administration for Market Regulation in 2023. However, our utilization of Eucommia leaf is still insufficient, and most of it is still regarded as waste, resulting in a serious waste of resources.

[0003] Chlorogenic acid is one of the main components of Eucommia ulmoides leaves. Due to its multiple natural activities, including regulating blood pressure, antiseptic, anti-tumor, anti-inflammatory and antioxidant properties, it has important value in the medical and food industries. Currently, several methods have been reported to be used for the recovery of chlorogenic acid from Eucommia ulmoides leaves. For example, Baoding Jizhong Pharmaceutical Co., Ltd. has disclosed a method for extracting chlorogenic acid from Eucommia ulmoides leaves (application number CN201810020306.1), and the steps mainly include: pulping of fresh leaves, ethanol soaking, pressing, sedimentation, recovery of ethanol, activated carbon decolorization, spray drying, etc.; Sichuan Jiuzhang Biochemical Technology Development Co., Ltd. has also disclosed a method for extracting chlorogenic acid from Eucommia ulmoides leaves (application number CN201410186187.9), and the steps mainly include: water extraction, pre- Treatment, macroporous resin adsorption, concentration and drying, etc.; Perfect (China) Co., Ltd. discloses a method for preparing chlorogenic acid from Eucommia ulmoides leaves (application number CN201110316708.4), the steps mainly include: enzymatic hydrolysis-assisted extraction, membrane separation, resin adsorption, etc.; Guangzhou Hanfang Modern Chinese Medicine Research and Development Co., Ltd. discloses a method for preparing high-purity chlorogenic acid and total flavonoids from Eucommia ulmoides leaves (application number CN200610123973.X), the steps mainly include: acidic ethanol solution extraction, macroporous resin adsorption, silica gel column separation and crystallization. Among the above invention patents, only the last one can obtain high-purity chlorogenic acid. However, the preparation process of this patent is complicated, and the extraction, macroporous resin adsorption, and silica gel column separation processes are time-consuming, labor-intensive, and solvent-consuming, and do not have cost advantages. Therefore, in order to meet the large demand for chlorogenic acid in the food, medicine, and cosmetics industries, it is urgent to develop a simpler and low-cost method for preparing chlorogenic acid in Eucommia ulmoides leaves. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a low-cost preparation method of high-purity chlorogenic acid in Eucommia ulmoides leaves.

[0005] The present invention provides a low-cost preparation method of high-purity chlorogenic acid in Eucommia ulmoides leaves, comprising the following process steps:

[0006] 1) Put the Eucommia leaf powder in an extraction container, add a certain amount of hydrochloric acid solution of a certain concentration and ethyl acetate, stir and extract at a certain speed for a certain time, stand or centrifuge, separate the organic layer, add a certain amount of ethyl acetate to the water layer after the first extraction, repeat the above extraction and separation operations several times, combine the obtained organic layers, and concentrate to dryness under reduced pressure to obtain TPSE extract;

[0007] 2) adding a certain amount of water to the TPSE extract, performing ultrasound-assisted extraction for a certain period of time at a certain ultrasonic power, centrifuging, and collecting the supernatant to obtain the USAE extract;

[0008] 3) Taking a certain amount of USAE extract, placing it on a reverse phase silica gel SPE column, adding a certain amount of ethanol solution of a certain concentration for elution, collecting the ethanol solution eluate, and concentrating it to dryness under reduced pressure to obtain the SPE extract (chlorogenic acid).

[0009] Preferably, the concentration of the hydrochloric acid solution used in step 1) is 0.2M.

[0010] Preferably, the amount (volume) of the hydrochloric acid solution used in step 1) is 5 times the amount (weight) of the Eucommia ulmoides leaves.

[0011] Preferably, the amount (volume) of ethyl acetate used in step 1) is 30 times the amount (weight) of Eucommia ulmoides leaves.

[0012] Preferably, the stirring speed used in step 1) is 1000 rpm.

[0013] Preferably, the stirring extraction time used in step 1) is 15 minutes.

[0014] Preferably, the extraction times used in step 1) is 3 times.

[0015] Preferably, the amount (volume) of water used in step 1) is 15 times the amount (weight) of Eucommia ulmoides leaves.

[0016] Preferably, the ultrasonic power used in step 2) is 500W.

[0017] Preferably, the ultrasound-assisted extraction time used in step 2) is 30 minutes.

[0018] Preferably, the amount of sample loaded (by weight) used in step 3) is equivalent to 1 times the amount of reversed-phase silica gel (by weight) (converted based on the weight of Eucommia ulmoides leaves).

[0019] Preferably, the concentration of the ethanol solution used in step 3) is 2.5%.

[0020] Preferably, the amount (volume) of the ethanol solution used in step 3) is 12 SPE column volumes.

[0021] The beneficial effects of the present invention are as follows: Compared with other existing methods, the present invention uses innovative integrated use of biphasic solvent extraction based on ethyl acetate, ultrasound-assisted extraction based on water, and solid phase extraction, which simplifies the entire process and greatly reduces the production cost, making it particularly suitable for large-scale preparation of high-purity chlorogenic acid in Eucommia ulmoides leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the method of the present invention.

[0023] Figure 2 It is the HPLC chromatogram of TPSE extract, USAE extract, SPE extract and 60% methanol in Example 1.

[0024] Figure 3 This is the mass spectrum of the SPE extract (chlorogenic acid) in Example 1 (negative ion mode).

[0025] Figure 4 This is the mass spectrum of the SPE extract (chlorogenic acid) in Example 1 (positive ion mode).

[0026] Figure 5 The hydrogen spectrum (CD 3 OD, 500MHz).

[0027] Figure 6 The carbon spectrum (CD 3 OD, 125MHz). DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific embodiments, but is not limited thereto.

[0029] Example 1

[0030] 1) Take the Eucommia leaf powder and place it in an extraction container, add 5 times (volume) of 0.2M hydrochloric acid solution of Eucommia leaf (weight) and 30 times (volume) of ethyl acetate of Eucommia leaf (weight), stir and extract at a speed of 1000 rpm for 15 minutes, centrifuge at 1000 rpm for 1 minute, and separate the organic layer. Add the same volume of ethyl acetate to the water layer after the first extraction, and repeat the above extraction operation twice. Combine the obtained organic layers, and concentrate to dryness under reduced pressure to obtain TPSE extract.

[0031] 2) Add 15 times the volume of water of Eucommia ulmoides leaves (weight) to the TPSE extract, perform ultrasound-assisted extraction for 60 min at an ultrasonic power of 500 W, centrifuge at 1000 rpm for 1 min, and collect the supernatant to obtain the USAE extract.

[0032] 3) Take an amount of USAE extract equivalent to 1 amount (weight) of reversed-phase silica gel (weight) (converted based on the weight of Eucommia ulmoides leaves), place it on a reversed-phase silica gel SPE column, add 12 SPE column volumes of 2.5% ethanol solution for elution, collect the ethanol solution eluate, and concentrate it to dryness under reduced pressure to obtain an SPE extract (total transfer rate of chlorogenic acid 80.9%, purity 98.4%).

[0033] Example 2

[0034] 1) Take Eucommia leaf powder and place it in an extraction container, add 0.2M hydrochloric acid solution of 5 times (volume) of Eucommia leaf (weight) and ethyl acetate of 40 times (volume) of Eucommia leaf (weight), stir and extract at a speed of 1250rpm for 30min, centrifuge at 1000rpm for 1min, and separate the organic layer. Add the same volume of ethyl acetate to the water layer after the first extraction, and repeat the above extraction operation 3 times. Combine the obtained organic layers, and concentrate to dryness under reduced pressure to obtain TPSE extract.

[0035] 2) Add 10 times the volume of water of Eucommia ulmoides leaves (weight) to the TPSE extract, perform ultrasound-assisted extraction for 30 min at an ultrasonic power of 400 W, centrifuge at 1000 rpm for 1 min, and collect the supernatant to obtain the USAE extract.

[0036] 3) Take the USAE extract equivalent to 0.75 times the amount (weight) of reversed-phase silica gel (weight) (converted based on the weight of Eucommia ulmoides leaves), place it on a reversed-phase silica gel SPE column, add 10 SPE column volumes of 2.5% ethanol solution for elution, collect the ethanol solution eluate, and concentrate it to dryness under reduced pressure to obtain the SPE extract (total transfer rate of chlorogenic acid 73.5%, purity 98.6%).

[0037] Example 3

[0038] 1) Take the Eucommia leaf powder and place it in an extraction container, add 0.1M hydrochloric acid solution of 10 times (volume) of Eucommia leaf (weight) and ethyl acetate of 50 times (volume) of Eucommia leaf (weight), stir and extract at a speed of 750 rpm for 60 minutes, centrifuge at 1000 rpm for 1 minute, and separate the organic layer. Add the same volume of ethyl acetate to the water layer after the first extraction, and repeat the above extraction operation twice. Combine the obtained organic layers, and concentrate to dryness under reduced pressure to obtain TPSE extract.

[0039] 2) Add 10 times the volume of water of Eucommia ulmoides leaves (weight) to the TPSE extract, perform ultrasound-assisted extraction for 60 min at an ultrasonic power of 350 W, centrifuge at 1000 rpm for 1 min, and collect the supernatant to obtain the USAE extract.

[0040] 3) Take the USAE extract equivalent to 0.5 times the weight of reversed-phase silica gel (weight) (converted to the weight of Eucommia ulmoides leaves), place it on a reversed-phase silica gel SPE column, add 14 SPE column volumes of 2.5% ethanol solution for elution, collect the ethanol solution eluate, and concentrate it to dryness under reduced pressure to obtain the SPE extract (the total transfer rate of chlorogenic acid is 87.0%, and the purity is 98.0%)

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention are included in the protection scope of the present invention.

Claims

1. A low-cost method for preparing high-purity chlorogenic acid from Eucommia ulmoides leaves, characterized in that The process steps include: 1) Put the Eucommia leaf powder in an extraction container, add a certain amount of hydrochloric acid solution of a certain concentration and ethyl acetate, stir and extract at a certain speed for a certain time, stand or centrifuge, separate the organic layer, add a certain amount of ethyl acetate to the water layer after the first extraction, repeat the above extraction and separation operations several times, combine the obtained organic layers, and concentrate to dryness under reduced pressure to obtain TPSE extract; 2) adding a certain amount of water to the TPSE extract, performing ultrasound-assisted extraction for a certain period of time at a certain ultrasonic power, centrifuging, and collecting the supernatant to obtain the USAE extract; 3) Taking a certain amount of USAE extract, placing it on a reverse phase silica gel SPE column, adding a certain amount of ethanol solution of a certain concentration for elution, collecting the ethanol solution eluate, and concentrating it to dryness under reduced pressure to obtain the SPE extract (chlorogenic acid).

2. The low-cost preparation method of high-purity chlorogenic acid in Eucommia ulmoides leaves according to claim 1, characterized in that: The concentration of the hydrochloric acid solution used in step 1) is 0.001-1M.

3. The low-cost preparation method of high-purity chlorogenic acid in Eucommia ulmoides leaves according to claim 1, characterized in that: The amount (volume) of the hydrochloric acid solution used in step 1) is 2.5-30 times the amount (weight) of the eucommia leaves.

4. The low-cost preparation method of high-purity chlorogenic acid in Eucommia ulmoides leaves according to claim 1, characterized in that: The amount (volume) of ethyl acetate used in step 1) is 10-50 times the amount (weight) of Eucommia ulmoides leaves.

5. The low-cost preparation method of high-purity chlorogenic acid in Eucommia ulmoides leaves according to claim 1, characterized in that: The stirring speed used in step 1) is 250-1500 rpm, and the stirring extraction time used in step 1) is 1-60 min.

6. The low-cost preparation method of high-purity chlorogenic acid in Eucommia ulmoides leaves according to claim 1, characterized in that: The extraction times used in step 1) are 1 to 5 times.

7. The low-cost preparation method of high-purity chlorogenic acid in Eucommia ulmoides leaves according to claim 1, characterized in that: The amount (volume) of water used in step 2) is 1 to 30 times the amount (weight) of the eucommia leaves.

8. The low-cost preparation method of high-purity chlorogenic acid in Eucommia ulmoides leaves according to claim 1, characterized in that: The ultrasonic power used in step 2) is 250-500W, and the ultrasonic-assisted extraction time used in step 2) is 5-120min.

9. The low-cost preparation method of high-purity chlorogenic acid in Eucommia ulmoides leaves according to claim 1, characterized in that: The weight of the sample amount used in step 3) (converted based on the weight of Eucommia ulmoides leaves) is equivalent to 0.1 to 1.5 times the weight of reversed-phase silica gel.

10. The low-cost preparation method of high-purity chlorogenic acid in Eucommia ulmoides leaves according to claim 1, characterized in that: The concentration of the ethanol solution used in step 3) is 0% to 10%, and the volume of the ethanol solution used in step 3) is 2 to 18 SPE column volumes.

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